Therapeutic compositions and methods for treating infections

ABSTRACT

This disclosure describes, in one aspect, a composition that includes a transgenic pathogen that expresses a heterologous pathogen associated molecular pattern (PAMP). In some embodiments, the pathogen may be attenuated. In some embodiments, the pathogen can include  T. cruzi . In another aspect, this disclosure describes a method of treating an infection in a subject. Generally, the method includes administering to the subject, in an amount effective to treat the infection, a pathogen genetically modified to express a pathogen-associated molecular pattern (PAMP) not natively expressed by the pathogen.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/754,127, filed Jan. 18, 2013, which is incorporated hereinby reference.

GOVERNMENT FUNDING

The present invention was made with government support under Grant No.AI44979 and Grant No. AI089952, each of which was awarded by TheNational Institutes of Health. The Government has certain rights in thisinvention.

SUMMARY

This disclosure describes, in one aspect, a composition that includes atransgenic pathogen that expresses a heterologous pathogen associatedmolecular pattern (PAMP) or associated molecular patterns (DAMP). Insome embodiments, the pathogen may be attenuated. In some embodiments,the PAMP can include an agonist of at least one Toll-Like Receptor (TLR)such as, for example, TLR 1, TLR 2, or TLR 5. In some embodiments, PAMPcan include an agonist of at least one intracellular pattern recognitionreceptor (PRR) such as, for example, Neuronal Apoptosis InhibitoryProtein 5/IL-1β-converting enzyme protease-activating factor(NAIP5/Ipaf).

In another aspect, this disclosure describes a method of treating aninfection in a subject. Generally, the method includes administering tothe subject, in an amount effective to treat the infection, a pathogengenetically modified to express a pathogen-associated molecular pattern(PAMP) not natively expressed by the pathogen. In some embodiments, thepathogen can include a virus, a bacterium, or a protozoan. In someembodiments, the protozoan can include T. cruzi. In some embodiments,the pathogen may be attenuated.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. PAMP transgenic T. cruzi secretes the PAMPs and presents it inall life cycle stages. (A) S. typhimurium flagellin (FliC) or N.meningitidis porin (PorB) cloned in pTREX expression vector with gp72signal sequence at their 5′ end to make transgenic T. cruzi secretingFliC (TcgFliC) or PorB (TcgPorB) (B) TcgFliC or Tcwt trypomastigotestage lysate and culture supernatant immunoblotted with antibodiesagainst FliC or α-tubulin. (C) FliC or PorB expression in theepimastigote, trypomastigote, and amastigote stages of TcgFliC orTcgPorB respectively determined by indirect immunofluorescence/DICmicroscopy, modifying the protocol described in the Examples. Anti-FliCor anti-HA antibodies were used to mark the target proteins.

FIG. 2. PAMP transgenesis in T. cruzi enhances cellular innate immuneresponses (A) NFkB/AP-1 activation in reporter cells incubated withwild-type T. cruzi (Tcwt), or PAMP transgenic T. cruzi (TcgFliC orTcgPorB) trypomastigotes for 12 hours. Media or E. coli-derived LPS wereused as negative and positive controls, respectively. (B) Caspaselactivation in (TLR 5) macrophages incubated with Tcwt or TcgFliCtrypomastigotes for 12 hours. Media or ATP with E. coli-derived LPSserved as negative and positive controls, respectively. (C) Theproportion of (TLR 5) macrophages producing IL-1β on incubation withTcwt or TcgFliC trypomastigotes for 18 hours. Media or ATP with E.coli-derived LPS served as negative and positive controls, respectively.(D) IL-1β mean fluorescence intensity (MFI) per cell by TLR 5⁻macrophages incubated with Tcwt or TcgFliC trypomastigotes for 18 hours.Media or ATP with E. coli-derived LPS served as negative and positivecontrols, respectively. All data show mean±SEM and are representative ofat least three separate experiments. * indicates p≦0.05, ** indicatesp≦0.01, in each case as determined by student t-test comparing theindicated groups to Tcwt.

FIG. 3. PAMP transgenesis in T. cruzi enhances the innate immuneresponses. NFkB/AP-1 activation in reporter cells incubated withepimastigote stage lysates of Tcwt, TcgFliC or TcgPorB for 18 hours.Media or E. coli-derived LPS served as negative and positive controls,respectively. Data represented as mean±SEM from one of three separateexperiments. * indicates p≦0.05, ** indicates p≦0.01, in each case asdetermined by student t-test comparing the indicated groups to Tcwt.

FIG. 4. PAMP transgenic T. cruzi induces higher frequencies of IL-12producing APCs (A) The proportion of peritoneal exudate macrophages withinduced IL-12 (YFP) on incubation with Tcwt, TcgFliC or TcgPorBtrypomastigotes for 18 hours. Media or E. coli-derived LPS served asnegative and positive controls, respectively. Data presented as mean±SEMand are representative of three separate experiments. (B) IL-12producing (CD11c⁺ CD8α⁺) cDC recruitment into the draining lymph nodeson Tcwt, TcgFliC or TcgPorB infection of IL-12yet40 mice. Flow plotsshow representative data from six days post-inoculation, with thenumbers inset indicating the percentage of IL-12 producing cDCs. (C)Kinetics of IL-12⁺ (CD11c⁺ CD8α⁺) cDC recruitment. Data are representedas mean±SEM from one of 3 separate experiments, with at least 3mice/group. * indicates p≦0.05, ** indicates p≦0.01, in each case asdetermined by student t-test comparing the indicated groups to Tcwt (A),at the corresponding time points (B).

FIG. 5. PAMP transgenesis in T. cruzi enhances systemic innate immuneresponses in vivo. (A) The proportion of blood derived monocytes andtheir IL-12 producing subset at the site (s.c) of Tcwt or TcgFliCinfection in IL-12yet40 mice at various time points post infection. Navemice were inoculated with media alone. (B) The proportion of residentmacrophages, CD11b⁺ DCs, or iDCs at the site of Tcwt or TcgFliCinfection in IL-12yet40 mice at various time points post infection. Navemice were inoculated with media alone. (C) Serum levels of variouscytokines in C57B1/6 mice infected with Tcwt or TcgFliC, four dayspost-inoculation. (D) Percentage of CD8⁺ T cells producing IFNγ in thedraining lymph nodes with Tcwt or TcgFliC infected (f.p) Yeti mice, sixdays post-inoculation. Representative flow plots with the numbers insetindicating the percentage of IFNγ producing CD8⁺ T cells. In the rightpanel, data represented as mean±SEM from one of three separateexperiments with 3-6 mice/group. * indicates p≦0.05, ** indicatesp≦0.01, in each case as determined by student t-test comparing theindicated groups to Tcwt (C and D), at the corresponding time points (Aand B).

FIG. 6. PAMP transgenesis in T. cruzi enhances the adaptive immuneresponses in mice. (A) Flow plots showing representative data from earlytime points post-infection in C57BL/6 mice with Tcwt or TcgFliC, withthe numbers inset indicating percentage of TSKb20⁺ CD8⁺ T cells. (B) Thekinetics of TSKb20⁺CD8⁺ T cell frequencies in circulation in TcgFliCcompared to Tcwt infection of C57BL/6 mice. Data represented as mean±SEMfrom one of six separate experiments, with at least six mice/group. (C)The kinetics of TSKb20⁺ CD8⁺ T cell frequencies in circulation inTcgPorB compared to Tcwt infection of C57BL/6 mice. Data represented asmean±SEM from one of six separate experiments, with at least sixmice/group. (D) The percentage of CD8⁺ (CD44⁺) T cells producing IFNγ(left) or TNFα (right) in response to TSKb20 peptide re-stimulation inTcwt, TcgFliC, or TcgPorB infection of C57BL/6 mice, 180 dayspost-inoculation. Data represented as mean±SEM from one of threeseparate experiments, with at least three mice/group. (E) The percentageof CD4’ (CD44⁺) T cells producing IFNγ (left) or TNFα (right) inresponse to T. cruzi whole cell lysate re-stimulation in Tcwt, TcgFliC,or TcgPorB infection in C57B1/6 mice, 180 days post-inoculation. Datarepresented as mean±SEM from one of three separate experiments, with atleast three mice/group. (F) Anti-T. cruzi antibody titers in sera ofmice infected with Tcwt or TcgFliC in C57B1/6 mice, 30 dayspost-inoculation. T. cruzi trypomastigote whole cell lysate served asthe antigen in the ELISA. Data shown as mean±SEM and is representativeof two separate experiments, with three mice/group. (G) The kinetics ofTSKb20⁺CD8⁺ T cell frequency in circulation on Tcwt, TcgFliC, or TcgPorBinfection of MyD88^(−/−) or C57B1/6 mice. Data represented as mean±SEMfrom one of three separate experiments, with 3-10 mice/group. *indicates p≦0.05, ** indicates p≦0.01, in each case as determined bystudent t-test comparing the indicated groups to Tcwt.

FIG. 7. Continuous expression of FliC is required to sustain theenhanced adaptive immunity. (A) The proportions IL-12 producing cDCsrecruited into the draining lymph nodes on various days after infectionwith T. cruzi having FliC temporarily surface-anchored (Tc-GPI-FliC),co-inoculated with (Tc-GPI+FliC), or constitutively expressed in(TcgFliC), compared to the background strain (Tc-GPI) in IL-12yet40reporter mice. The flow panel shows representative flow plots, with thenumbers inset indicating the % IL-12⁺ cDCs. Data are represented asmean±SEM from one of three separate experiments, with at least threemice/group/time point. (B) TSKb20⁺CD8⁺ T cell frequency in circulationon Tc-GPI, Tc-GPI-FliC, Tc-GPI+FliC or TcgFliC infection of C57BL/6mice. Data represented as mean±SEM from one of three separateexperiments, with three mice/group. * indicates p≦0.05, ** indicatesp≦0.01, in each case as determined by student t-test comparing theindicated groups to Tc-GPI at the corresponding time points.

FIG. 8. Continuous expression of FliC sustains the enhanced adaptiveimmunity. (A) Percentage of T. cruzi retaining the GPI-biotin at varioustime points post surface-anchoring, when maintained at 37° C. asdetermined by flow cytometry. Data are representative of two separatetrials. (B) NFkB/AP-1 activation in reporter cells incubated withTc-GPI, Tc-GPI-FliC, or TcgFliC trypomastigotes for 12 hours. Media orE. coli-derived LPS served as negative and positive controls,respectively. Data represented as mean±SEM from one trial. * indicatesp<0.05 as determined by student t-test comparing the indicated groups toTc-GPI. (C) The proportion of peritoneal exudate macrophages withinduced IL-12 production on incubation with Tcwt, Tc-GPI-FliC, orTcgFliC trypomastigotes for 18 hours. Media or E. coli-derived LPSserved as negative and positive controls, respectively. Data representedas mean±SEM from two separate trials. * indicates p≦0.05 as determinedby student t-test comparing the indicated groups to Tcwt. (D) Thepercentage of IL-12 producing cDCs recruited into the draining lymphnodes observed on various days after infection with T. cruzi having theindicated PAMPs temporarily anchored on, co-inoculated with, orconstitutively expressed (TcgFliC), compared to the background strain(Tc-GPI), in IL-12yet40 reporter mice. Data are represented as mean±SEMfrom one of two separate experiments, with at least threemice/group/time point. (E) The TSKb20⁺CD8⁺ T cell frequency incirculation on infection with T. cruzi having the indicated PAMPstemporarily anchored on, co-inoculated with, or heterologouslyover-expressed (TcgFliC), compared to the background strain (Tc-GPI) inC57BL/6 mice, 240 days post infection. Data represented as mean±SEM fromone of three separate experiments, with three mice/group. In (D) and(E), * indicates p≦0.05, ** indicates p≦0.01, in each case as determinedby student t-test comparing the indicated groups to Tc-GPI, at thecorresponding time points.

FIG. 9. FliC transgenesis enhances control of T. cruzi infection in miceas indicated by an increase in central memory phenotype T cells andreduced parasite load. (A) Frequency of CD8⁺ T cells in circulationhaving Tcm like phenotype (CD127^(hi) TSKb20⁺ CD8⁺ (left) and KLRG1^(lo)CD44⁺ CD8⁺ (right)), in Tcwt or TcgFliC infected C57BL/6 mice, 296 dayspost-inoculation. Data represents three separate experiments with 3-6mice/group. (B) T. cruzi DNA in skeletal muscle of C57BL/6 miceinoculated with Tcwt or TcgFliC as determined by quantitative real-timePCR, 400 days post-inoculation. Horizontal bars represent the mean.Naïve mice served as control. The dotted line represents the thresholdof detection for the assay. Data are representative of five separateexperiments with at least six mice/group. * indicates p≦0.05, **indicates p≦0.01, and n.s indicates p>0.05, in each case as determinedby student t-test.

FIG. 10. (A) PAMP transgenesis does not influence the virulence of T.cruzi. (A) T. cruzi trypomastigotes in circulation observed inTcwt-infected or TcgFliC-infected IFNγ^(−/−) mice, 21 dayspost-inoculation. Data are represented as mean±SEM from one of twoseparate experiments, with at least three mice/group. (B) Mortalityobserved in Tcwt or TcgFliC infected IFNγ^(−/−) mice. Datarepresentative of at least two separate experiments with threemice/group. n.s indicates p>0.05, as determined by student t-test.

FIG. 11. Inoculation of previously infected mice with TcgFliC parasitesmarkedly boosts the T. cruzi-specific T cell response. Mice chronicallyinfected with wild-type T. cruzi (>300 days post-inoculation) wererechallenged with 10⁴ TcgFliC, TcgPorB, or Tcwt trypomastigotes in thefoot-pad. The level of T. cruzi-specific (TSKb20⁺) CD8⁺ T cells was thenmeasured. The mice challenged with Tcwt had a brief and modestenhancement in T. cruzi-specific T cell responses but mice receivingeither TcgFliC or TcgPorB experienced a threefold to fivefold increasein response that persisted for at least 100 days. Data represents one ofthree separate experiments.

FIG. 12. Mice “vaccinated” with TcgFliC parasites show an increase inCD127 expression on their T. cruzi-specific CD8+ T cells, indicative ofthe relative absence of antigen stimulation and thus an enhancedclearance of parasites in the mice boosted with PAMP-transgenic T.cruzi. Mice chronically infected with wild-type T. cruzi (>300 dayspost-inoculation) were rechallenged with 10⁴ TcgFliC, TcgPorB, or Tcwttrypomastigotes in the foot-pad and the TSKb20⁺ CD8⁺ T cell populationwas analyzed for the expression the central memory T cell marker CD127.The increase in CD127 expression is indicative of the relative absenceof antigen stimulation and thus an enhanced clearance of parasites inthe mice boosted with PAMP-transgenic T. cruzi. Data represents one ofthree separate experiments.

FIG. 13. Mice “vaccinated” with TcgFliC parasites show a decrease KLRG1expression on their T. cruzi-specific CD8+ T cells, indicative of adecrease in antigen stimulation and thus an enhanced clearance ofparasites in the mice boosted with PAMP-transgenic T. cruzi. Micechronically infected with wild-type T. cruzi (>300 dayspost-inoculation) were rechallenged with 10⁴ TcgFliC, TcgPorB, or Tcwttrypomastigotes in the foot-pad and the TSKb20⁺ CD8⁺ T cell populationwas analyzed for the expression the effector T cell marker KLRG1. Thedecrease in KLRG1 expression is indicative of the relative absence ofantigen stimulation and thus an enhanced clearance of parasites in themice boosted with PAMP-transgenic T. cruzi. Data represents one of threeseparate experiments.

FIG. 14. Change in T. cruzi-specific T cell response expressed asfold-increase following Tcwt or TcgFliC (flagellin-expressing T. cruzi)challenge. Data represents one of four separate experiments.

FIG. 15. In contrast to wild-type parasites expressing FliC (TcgFliC)that may persist for a long-term in mice, T. cruzi deficient in a geneencoding a protein of unknown function (KO-10) have much reducedvirulence and limited persistence in mice. As a consequence, KO-10parasites also induce a much reduced T. cruzi-specific T cell response,which is not enhanced by the expression of either FliC or PorB. Datarepresents one of two separate experiments.

FIG. 16. PAMP transgenic KO10 parasites also appear to be more readilycontrolled than either wild-type or KO-10 parasites, as suggested byincreased central memory markers (CD127 and CD62L) and reducedactivation markers (KLRG1) on T. cruzi-specific (TSKb20⁺ CD8⁺) T cells

FIG. 17. KO-10/KO-10gFliC parasites delivered as a therapeutic vaccineinduce enhanced control of an ongoing infection (520 days postinfection, at time of vaccination) as indicated by the much reduced tonegligible parasite tissue load detected 230 days post vaccination.Horizontal bars represent the mean. Not vaccinated (Unrechallenged) miceserved as controls. The dotted line represents the threshold ofdetection for the assay. Data are representative of three separateexperiments with at least 3-6 mice/group. The indicated p values weredetermined by student t-test.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

This disclosure describes compositions and methods useful for treatinginfections by, for example, bacteria, viruses, and parasites. One cangenetically modify a pathogen such as, for example, the human parasiteT. cruzi to express one or more pathogen associated molecular patterns(PAMPs) that are not natively expressed by the pathogen. PAMPs areinvolved in both innate immunity and adaptive immunity. Administering apathogen genetically modified to express one or more PAMPs can augmentthe subject's native immune response to the infection. As a result,administering the genetically modified pathogen as described herein canresult in a reduction of the duration, extent, and/or severity of theinfection. Moreover, if one selects a pathogen such as T. cruzi that canpersist in the subject after administration, the beneficial effects ofthe PAMP expression by the pathogen can similarly persist for a periodgreater than by, for example, simply providing free PAMPs as an adjuvantcomponent in a vaccination or other medicament.

As used herein, the term “treat” or variations thereof refer toreducing, limiting progression, ameliorating, or resolving, to anyextent, the symptoms or clinical signs related to a condition. As usedherein, “ameliorate” and variations thereof refer to any reduction inthe extent, severity, frequency, and/or likelihood of a symptom orclinical sign characteristic of a particular condition. “Symptom” refersto any subjective evidence of disease or of a patient's condition.“Sign” or “clinical sign” refers to an objective physical findingrelating to a particular condition capable of being found by one otherthan the patient.

In the description that follows, the term “and/or” means one or all ofthe listed elements or a combination of any two or more of the listedelements; the terms “comprises” and variations thereof do not have alimiting meaning where these terms appear in the description and claims;unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one; and the recitationsof numerical ranges by endpoints include all numbers subsumed withinthat range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

Pathogen associated molecular patterns (PAMPs) are known to be involvedin initiating innate immune responses and to inducing and directingsubsequent adaptive immunity. PAMPs are effective indicators of thepresence of particular pathogens, in part, because certain PAMPs can beunique to certain classes of pathogens. Also, certain PAMPs can berequired for pathogen survival and thus cannot be easily altered,suppressed, or hidden by a pathogen. Thus, most of the studies thatestablished the role of PAMPs in inducing and directing innate oradaptive immune responses are based on negating the ability of the hostto respond to these molecular patterns rather than by blocking PAMPexpression.

We use the protozoan Trypanosoma cruzi as a model pathogen to evaluatethe role of PAMPs in influencing adaptive immunity at and beyond itsonset. As used herein, the term “pathogen” refers to any conventionalinfectious pathogen (e.g., a bacterium, a virus, a fungus, a protozoan,a helminth, etc.) but also includes non-infectious cells such as, forexample, tumor cells. Thus, a pathogen may be acellular, unicellular, ormulticellular. While described below in the context of an exemplaryembodiment in which the pathogen is T. cruzi, the compositions andmethods described herein can be made and/or practiced using anypathogen. Exemplary alternative pathogens include, for example,infectious pathogens such as, for example, a bacterium, a virus, afungus, a protozoan, or a helminth, as well as non-infectious pathogenssuch as, for example, tumor cells.

T. cruzi is the etiological agent of Chagas disease, a prevalent humanparasitic disease in the Americas. T. cruzi trypomastigotes stimulate avery weak host cell response during invasion and elicit significantlydelayed adaptive immune responses, strongly suggesting the relativeabsence of potent PAMPs on live, invading parasites. Although severalendogenous PAMPs have been identified in T. cruzi, their failure toimpact the strength of adaptive immune responses suggests that theputative PAMPs are not readily available on live T. cruzi and havelittle relevance to anti-T. cruzi immunity.

Specifically, we generate transgenic T. cruzi expressing potentexogenous (non-T. cruzi, bacteria-derived) protein PAMPs and show thatthis expression induces superior innate immune responses and drives morerapid and persistently stronger adaptive immunity in mice. Expression ofbacterial PAMPs by transgenic T. cruzi resulted in enhanced innateimmune responses and a more robust T. cruzi-specific CD8⁺ T cellresponse, with increased IFNγ- and TNFα-producing CD4⁺ and CD8⁺ T cells.Co-inoculating PAMPs with T. cruzi or temporarily anchoring exogenousPAMPs on T. cruzi can enhance the early adaptive immune response. Ourtransgenic T. cruzi that can continuously express PAMPs, however, wereable to sustain this enhanced response and thus promote better controlof the infection. T. cruzi transgenic for bacterial PAMP expression alsoboost T. cruzi-specific immune responses in mice chronically infectedwith wild-type T. cruzi and reduce parasite load in these mice, thusdemonstrating their potential as therapeutic vaccines. These findingsfurther support the relevance of PAMPs, particularly in persistentinfections, and may also be applicable for improving live-attenuatedvaccines.

PAMP Transgenesis in T. cruzi Enhances Innate Immune Responses

We chose to express protein PAMPs in T. cruzi since PAMP expression canbe stably generated by transgenesis of a single coding region. Incontrast, polysaccharides or nucleic acid PAMPs require transferring anentire biosynthetic pathways into T. cruzi. Coding regions encoding theSalmonella typhimurium flagellin (fliC) and Neisseria meningitidis porin(porB) were amplified by PCR and cloned into the pTREX plasmid (Lorenziet al., 2003. Gene 310:91-99) with a T. cruzi secretory signal peptidefrom gp72 (Garg et al., 1997. J Immunol 158:3293-3302) at their 5′ end(FIG. 1A). S. typhimurium FliC is a ligand for both TLR 5 and NeuronalApoptosis Inhibitory Protein (NAIP)5/IL-1β-converting enzymeprotease-activating factor (Ipaf). Neisseria meningitidis PorB is aligand for TLR 1/2. PAMP-transgenic T. cruzi expressing FliC (TcgFliC)or PorB (TcgPorB) were engineered by transfecting these constructs intowild-type (WT), Brazil strain T. cruzi (Tcwt). The signal peptideensured secretion (FIG. 1B) of the protein PAMPs expressed by thePAMP-transgenic T. cruzi in epimastigote, trypomastigote, and amastigotelife stages (FIG. 1C).

Stimulation of TLRs 1/2 or TLR 5 ultimately activates the transcriptionfactors NFkB/AP-1 and promotes immunity at least in part by inducing theproduction of inflammatory cytokines NFkB/AP-1 reporter cell linesexhibited significantly increased NFkB/AP-1 activation by TcgFliC orTcgPorB live trypomastigotes (FIG. 2A) or epimastigote lysates (FIG. 3)relative to wild-type T. cruzi parasites (Tcwt). FliC is also anNAIP5/ipaf ligand that induces IL-1β production in antigen presentingcells (APCs). FliC-expressing T. cruzi potentiated strong caspaselactivation (FIG. 2B) and production of IL1β in TLR 5-deficientmacrophages (FIGS. 2C and 2D), demonstrating that T. cruzi-expressedFliC exhibits both of the PAMP properties of Salmonella flagellin.

The innate immune response-inducing activity of PAMP-transgenic T. cruzitrypomastigotes was also evident in IL-12yet40 reporter mice in whichcells expressing IL-12/IL-23 p40 subunit also express yellow fluorescentprotein (YFP). Peritoneal exudate macrophages exposed in vitro toPAMP-transgenic T. cruzi produced IL-12 at an increased frequencyrelative to those exposed to wild-type T. cruzi (FIG. 4A). Additionally,TcgFliC and TcgPorB infections of IL-12yet40 reporter mice resulted in amore rapid and increased infiltration of the IL-12-producing CD11c⁺CD8α⁺ classical dendritic cells (cDCs) into the draining lymph nodes(FIGS. 4B and 4C). TcgFliC infection also altered the lineage bias ofthe inflammatory cells infiltrating or prevailing at the site ofinfection, with increased numbers of blood-derived monocytes(CD45⁺CD11b⁺CD11c⁻Gr-1^(int)), macrophages (CD45⁺CD11b⁺CD11c⁻F4/80⁺),inflammatory DCs (CD45⁺CD11b⁺CD11c^(hi)Gr-1^(int)), and othernon-classical (CD45⁺CD11b⁺CD11c⁺CD8α⁻) DCs, as compared to wild-type T.cruzi infection (FIGS. 5A and 5B). Classical (CD45⁺CD11b⁻CD11c⁺CD8α⁺)DCs remained undetectable at the site of infection in either case.TcgFliC infection was also associated with enhanced recruitment ofIL-12-producing monocytes (FIG. 5A) and neutrophils(CD45⁺CD11b⁺Gr-1^(hi)) to the site of infection. The innate immuneenhancing effect of PAMP transgenesis was also evident systemically withsignificantly higher serum levels of IL-12 and TNFα compared to Tcwtinfection (FIG. 5C).

IFNγ produced by naïve CD8⁺ T cells in a T cell receptor(TCR)-independent, IL-12-mediated manner early in the infection appearsto be involved in the initial immune responses to a number of pathogens.To measure the IFNγ induced early in response to T. cruzi infection, weused the IFNγ reporter (Yeti) mice, wherein cells expressing IFNγconcurrently express enhanced-yellow fluorescent protein (eYFP) (Mayeret al., 2005. J Immunol 174:7732-7739; Stetson et al., 2003. J Exp Med198:1069-1076). We observed significantly higher proportions ofIFNγ⁺CD8⁺ T cells (but undetectable T. cruzi-specific (TSKb20⁺) CD8⁺ Tcells (not shown)) in the draining lymph nodes with TcgFliC infectioncompared to infection with wild-type T. cruzi, Tcwt (FIG. 5D). Takentogether, these results indicate that the expression of bacterial PAMPsin T. cruzi markedly enhances innate immune activation both in vitro andin vivo.

PAMP Transgenesis in T. cruzi Enhances Adaptive Immune Responses

The control of T. cruzi infection in mice can involve a robust T.cruzi-specific CD8⁺ T cell response. The CD8⁺ T cell response to T.cruzi in C57BL/6 mice is dominated by cells specific for peptidesencoded by the trans-sialidase gene family (Martin et al., 2006. PLoSPathog 2:e77). Hence, we can use TSKb20⁺CD8⁺ T cells as a surrogate forthe total CD8⁺ T cell response mounted against T. cruzi, and track thisresponse using the TSKb20/K^(b) tetramers (Martin et al., 2006. PLoSPathog 2:e77). Mice infected with PAMP-transgenic T. cruzi mounted amore rapid (FIG. 6A) and significantly stronger TSKb20⁺ CD8⁺ T cellresponse that was also maintained at higher levels throughout theinfection (FIGS. 6B and 6C) relative to wild-type infected mice. Thispotentiation of T cell responses by PAMP-transgenic T. cruzi was alsoevident in the IFNγ and TNFα production by antigen-experienced CD8⁺(FIG. 6D) and CD4⁺ (FIG. 6E) T cells. Infection with PAMP-transgenic T.cruzi also elicited higher serum levels of T. cruzi specific antibodiescompared to infection with Tcwt parasites (FIG. 6F).

To reaffirm that the enhanced adaptive immune responses observed withPAMP transgenesis in T. cruzi were indeed dependent on signaling throughpattern recognition receptors (PRRs) targeted by the transgenic PAMPs,we infected MyD88^(−/−) mice, which are deficient in the primary adaptorfor multiple TLRs and are unresponsive to TLR 5, TLR 1/2, or (IL-1βfrom) NAIP5/ipaf stimulation. The T. cruzi-infected MyD88^(−/−) miceshowed a delayed generation of TSKb20-specific T cells relative towild-type mice with the pattern of responses being similar regardless ofexpression of the bacterial PAMPs (FIG. 6G). This result indicates thatthe enhanced adaptive immune response to T. cruzi conferred by PAMPtransgenesis is a result of increased triggering of host PRRs and theconsequential effects downstream of MyD88 signaling.

Continuous Expression of FliC is Required to Sustain the EnhancedAdaptive Immunity

A canonical concept in immunology is that strong innate immunity invokesmore potent adaptive immune responses. This concept is supported by manystudies demonstrating that co-delivery of TLR-ligands with antigens orvaccines significantly boosts adaptive immune responses. However, to ourknowledge, no studies have directly investigated the impact on adaptiveimmunity of a transient expression of PAMPS at the initiation ofinfection compared to a continuous presence of PAMPs throughout thecourse of infection. Given that PAMP-transgenesis in T. cruzi not onlyinitiated a more rapid TSKb20⁺CD8⁺ T cell response in mice but alsoresulted in a response that was maintained at unusually high levels,PAMPs may have a continuous instructive role in maintaining strongadaptive immune responses.

To determine the consequences of transient versus continuous expressionof PAMPs on adaptive immune responses to T. cruzi, we tethered variousPAMPs to T. cruzi using GPI-anchors. Initial experiments showed thatmolecules linked in this fashion were readily incorporated into thesurface of trypomastigotes of T. cruzi and had a half-life ofapproximately 12 hours (FIG. 8A). The signaling potency of FliCdelivered by the GPI tether (Tc-GPI-FliC) or by endogenous expression(TcgFliC) was equivalent, as indicated by their similar abilities toinduce NFkb/AP-1 activation in reporter cells (FIG. 10B) or IL-12production in peritoneal exudate macrophages (FIG. 8C). Additionally,Tc-GPI-FliC, TcgFliC, or native FliC co-inoculated with T. cruzi allpotentiated similar innate immune responses to T. cruzi in vivo (FIG.7A), and resulted in nearly identical peak TSKb20⁺CD8⁺ T cells responses(FIG. 7B). Only in the infection with TcgFliC, however, was theTSKb20-specific response maintained above the level of the Tcwtinfection into the chronic stage (FIG. 5B). The delivery of otherindividual or combinations of PAMPs with T. cruzi infection byGPI-anchors or by co-inoculation enhanced innate (FIG. 8D) immuneresponses in mice—some much more strongly than TcgFliC. But onlyinfection with the PAMP-transgenic T. cruzi resulted in the long-termmaintenance of the enhanced adaptive responses (FIG. 8E). Thus,continued expression of PAMPs acts to maintain stronger adaptive immuneresponses, exceeding those elicited by transient PRR engagement at theinitiation of infection.

FliC Transgenesis Enhances Control of T. cruzi Infection in Mice

To investigate the impact of the PAMP-induced enhancement of innate andadaptive immune responses on the parasite control during the course ofT. cruzi infection, we first monitored the phenotype of T.cruzi-specific CD8⁺ T cells in these mice. Drug-induced cure of T. cruziinfection results in a gradual shift in the TSKb20⁺CD8⁺ T cells from apredominant T-effector phenotype)(CD127^(lo)) to a majority T-centralmemory (T_(CM))-like (CD127^(hi)) phenotype, accompanied by a decreasein the frequency of CD8⁺ T cells expressing KLRG1, a marker for repeatedantigenic stimulation.

At 296 days post-inoculation, TcgFliC-infected mice exhibited higherproportions of T_(cm) among the TSKb20⁺CD8⁺ T cells and decreasednumbers of KLRG1⁺(CD44⁺)CD8⁺ T cells in comparison to mice infected withwild-type T. cruzi (FIG. 9A), suggesting more effective control of theinfection with TcgFliC. This conclusion was confirmed using qPCR tomeasure T. cruzi DNA in muscle tissue from these mice. At 400 dayspost-inoculation, T. cruzi DNA was undetectable in mice infected withTcgFliC, but was consistently detected in tissues from Tcwt infectedmice (FIG. 9B).

Immunosuppression can reveal otherwise undetectable infection and be adefinitive measure of drug-induced cure in T. cruzi infection. One ofthree TcgFliC-infected mice immunosuppressed with cyclophosphamideexhibited no detectable parasites after immunosuppression, indicatingclearance of the infection (data not shown). The enhanced control of theTcgFliC infection relative to wild-type T. cruzi infection was not dueto a decrease in virulence of the FliC-transgenic parasites, asIFNγ^(−/−) mice infected with TcgFliC or Tcwt showed similar peripheralblood parasite loads and mortality patterns (FIG. 10). Taken together,these data indicate that FliC transgenesis potentiates adaptive immuneresponses and facilitates control and clearance of T. cruzi infection.

One paradigm in immunology is that innate immune mechanisms detectmicrobial infections through their characteristic PAMPs and trigger thespecific antimicrobial host defense responses appropriate to thatinfection. Once initiated, these pathogen-specific adaptive immuneresponses bring about control of the infection and, often, a long-termspecific immunological memory. However, we have very limited knowledgeof the role of PAMPs in influencing the adaptive immunity beyond itsinitiation, in large part because, by their very nature, PAMPS arecrucial for pathogen survival and thus cannot be turned off during aninfection. In this study we provide unequivocal evidence that theexpression of classical bacterial PAMPS in the protozoan pathogen T.cruzi results in substantially enhanced innate and adaptive immuneresponses and more efficient pathogen control. These data add to thewealth of information indicating that T. cruzi has an extremely quietentry into hosts and a considerably delayed induction of anti-parasiticimmune responses.

Though PAMPs are highly conserved structures that are extremelydifficult for pathogens to alter or sacrifice, there is some plasticityin PAMP display. For example, host detection of LPS in Porphyromonasgingivalis and Escherichia coli is modulated by differential acylationof lipid-A, while Yersinia pestis synthesizes LPS-lipid A that is a poorTLR 4 ligand. Helicobacter pylori produces a flagellin that isnon-stimulatory to TLR 5 and Pseudomonas aeruginosa down-regulates itsflagellin expression in airway passages. Although it is unlikely thatany pathogen will be able to make all its PAMPs entirely invisible tothe immune system, potential PAMPs could be rendered immunologicallyinconsequential by concealing or modifying the PAMP withoutsignificantly impacting pathogen biology.

Multiple PAMPs (e.g., GPI anchors, T. cruzi DNA, GIPL-ceramide) havebeen attributed to T. cruzi but these molecules seem to be relativelyinsignificant to the downstream immune responses generated. The apparentinsignificance of T. cruzi PAMPs may be due, at least in part, to theseputative PAMPs being “hidden” from their respective TLRs in live, intactT. cruzi. However, when strong bacterial PAMPs are transgenicallyexpressed and released by T. cruzi, significantly improved innate andadaptive immune responses are generated. Thus, the failure of T. cruzito display potent PAMPs may indeed be another example of innate immuneevasion employed by pathogens.

The observed evasion of innate immune responses may not only beimportant in delaying the adaptive immune response (thus allowing forfirm establishment of the infection), but also may promote thepersistence of T. cruzi. In the presence of a bacterial PAMP, T. cruziinfection is better controlled and even completely cleared in somecases. Complete clearance of T. cruzi infection is normally extremelyrare. Given the increased level of T. cruzi-specific CD8⁺ T cells with aTcm phenotype and the nearly undetectable tissue parasite load in miceinfected with PAMP transgenic T. cruzi, most of these mice wouldeventually cure these infections if allowed sufficient time.Importantly, this enhanced control of T. cruzi infection derived fromexpression of bacterial PAMPs is not associated with any evidence ofincreased immunopathology.

The relative absence of PAMPs in T. cruzi provided a unique opportunityto study the importance of PAMP expression beyond the early induction ofadaptive immune responses. Though there is a wealth of literaturedemonstrating how the strength of innate immunity determines the potencyof adaptive immune responses, few studies have focused on the impact ofinnate immune responses on adaptive immunity once an infection isestablished. When potent bacterial PAMPs were either co-inoculated with,temporarily surface-anchored on, or constitutively expressed by theinvading T. cruzi, the resulting adaptive immune responses were not onlyaccelerated, but also peaked at levels that were significantly above thelevels seen in mice infected with wild-type T. cruzi. It was only whenT. cruzi perpetually expressed the PAMPs, however, that the strongeradaptive immune responses were maintained throughout the course of theinfection, eventually leading to a better control of the pathogen andsterile clearance in some cases.

Without wishing to be bound by any particular theory, the quality,quantity, and longevity of T and B cells may be potentiated by thegenerally enhanced inflammatory milieu and/or the improved antigenprocessing and presentation by more highly activated APCs resulting fromcontinuing PAMP exposure. Transgenic expression of FliC and PorB inducedsimilar boosting of immune responses. Moreover, transgenic expression ofTcgFliC further corresponded with better parasite control. FliC isdistinctive in its ability to induce IL-1β through the intracellularNAIP5/ipaf receptor stimulation. Given that T. cruzi spends the majorityof its time in vertebrates in the cytoplasm of host cells that arelikely to express the NAIP5/ipaf receptor, it is possible thatNAIP5/ipaf-IL-1β activation contributes to enhanced recognition andcontrol of TcgFliC-infected cells. IL-1β levels have also been shown tocorrelate with CD8⁺ T cell abundance in adipose tissue, which is a majordepot for chronic T. cruzi persistence.

A possible confounder in the interpretation of our results is that FliCexpressed by TcgFliC may act as a target antigen for adaptive immuneresponses, contributing to the control of FliC-expressing parasites.However, the absence of detectable FliC-specific CD4⁺ or CD8⁺ T cells inTcgFliC-infected mice argues against this possibility. Additionally, T.cruzi expressing the highly immunogenic chicken ovalbumin (OVA) proteinthat induces very strong OVA-specific T cells does not appear to becontrolled any better than wild-type T. cruzi, further suggestingagainst control of T. cruzi infection through an adaptive response to atarget antigen.

The deficiency of effective PAMPs in T. cruzi may be only one of theseveral factors that contribute to the marked delay in initiation ofanti-T. cruzi immune responses. An additional trigger for induction ofadaptive responses is exposure of damage associated molecular patterns(DAMPs). Revelation of DAMPs from either host or T. cruzi would not beexpected until 4-5 days post-infection, with the initial round of exitof T. cruzi from infected host cells.

Thus, innate immune responses may have an extended instructive role onadaptive immunity, thus playing an even more significant part in theeffective control of pathogens than was previously appreciated. Theinability of classical adjuvants to productively stimulate innateimmunity and to generate long-lasting T cell responses has been a hurdlein the development of T-cell-based vaccines. Our observation that PAMPtransgenesis generates stronger and longer lasting specific immunity mayaid in the development of better vaccination strategies, especially oflive attenuated vaccines.

For example, rechallenge of chronically infected mice withPAMP-expressing (either flagellin-expressing (TcgFliC) orporin-expressing (TcgPorB)) transgenic T cruzi elicited a stronger, moresustained recall immune response. (FIG. 11 and FIG. 14). Moreover, FIG.12 shows an increase in the proportion of CD127⁺TSKb20⁺ CD8⁺ T cells inmice rechallenged with PAMP-transgenic T. cruzi compared toun-rechallenged mice or mice rechallenged with wild-type T. cruzi. Asdescribed earlier, the higher proportion of CD127⁺TSKb20⁺ CD8⁺ T cellsindicate better control or clearance of T. cruzi infection. FIG. 13shows a decrease in the proportion of KLRG1⁺TSKb20⁺ CD8⁺ T cells in micerechallenged with PAMP-transgenic T. cruzi compared to un-rechallengedmice or mice rechallenged with wild-type T. cruzi. The decrease in theproportion of KLRG1⁺TSKb20⁺ CD8⁺ T cells also may be considered anindicator of progressive clearance of T. cruzi infection.

T. cruzi deficient in a gene encoding a protein of unknown function(KO10) have much reduced virulence and limited persistence in mice. As aconsequence, KO10 parasites also induce a much reduced T. cruzi-specificT cell response which is not enhanced by the expression of either FliC(KO10gFliC) or PorB (KO10gPorB) (FIG. 15). However, PAMP transgenic KO10parasites appear to be more readily controlled than either Tcwt or KO10parasites, as suggested by increased central memory markers (CD127 andCD62L) and reduced activation markers (KLRG1) on T. cruzi-specific(TSKb20⁺ CD8⁺) T cells (FIG. 16). As a consequence of the recall immuneresponse generated, rechallenging chronically infected mice with KO10,KO10gFliC or KO10gPorB induced a better control of T. cruzi.KO10/KO10gFliC parasites delivered as a therapeutic vaccine inducedenhanced control of an on-going infection (520 days at time ofvaccination) as indicated by the much reduced to negligible parasitetissue load detected 230 days post vaccination (FIG. 17), indicating thepotential of therapeutic live-vaccinations in persistent infections.

Thus, in one aspect, this disclosure describes a composition thatincludes a transgenic pathogen (e.g., an infectious pathogen or othercell such as, for example, a tumor cell) that expresses a heterologouspathogen associated molecular pattern (PAMP) or a heterologous damageassociated molecular patterns (DAMP). As used herein, “heterologous”refers to a PAMP that is not natively produced by the pathogen.

The compositions described herein can be employed in connection with any“live vaccine” (e.g., polio or live vaccine vector (vaccinia,cytomegalovirus, adenovirus, etc.)). Most vaccines administered tohumans are not given as live vaccines for safety reasons. Some, however,may be made safer and more acceptable with PAMP expression. Moreover,the compositions described herein may be employed in connection withlive vaccines administered to animals including, for example, livestockand/or companion animals. In an individual with a persistentinfection—i.e., the individual is already infected by apathogen—therapeutic administration of a composition that includes alive pathogen genetically modified as described herein can amplify theindividual's existing immune response to the infecting pathogen.

The PAMP can be any PAMP that can induce an innate immune response to apathogen that expresses the PAMP, acting through pattern recognitionreceptors (PRRs) on host cells. Exemplary PAMPs include, but are notlimited to, Toll-like receptor (TLR), Nod-like receptor (NLR), C-typelectin receptor (CLR) or RIG-I-like receptor (RLR) ligands. SuitableTLRs include, for example, TLR 1, TLR 2, TLR 3 TLR 4, TLR 5, TLR 6, TLR7, TLR 8, TLR 9, TLR 10, TLR 11, TLR 12, and TLR 13, Suitable NLRsinclude, for example, Neuronal Apoptosis Inhibitory Protein5/IL-1β-converting enzyme protease-activating factor (NAIP5/Ipaf) orNACHT, LRR and PYD domains-containing protein 3 (NALP3). Suitable RLRsinclude Retinoic acid-inducible gene 1 (RIG-I), MelanomaDifferentiation-Associated protein 5 (MDA5) or RIG-I-like receptor 3(LGP2). Suitable CLRs include Dec205, Dectin-1, Dectin-2, DNGR-1, etc.

The DAMP can be any DAMP that can induce an innate immune response to acell that expresses the DAMP. Exemplary DAMPs include, but are notlimited to DNGR-1, or HMGB1 receptor(s).

In some embodiments, the cell expressing the PAMP or DAMP may be acancer cell or an infectious pathogen—e.g., T. cruzi. In such cases, thepathogen may be attenuated. Such embodiments can have utility as atherapeutic or prophylactic agent that may be administered to a subjecthaving, or at risk of having, an infection of a non-attenuated form ofthe pathogen. As used herein, a subject having a condition caused byinfection by the pathogen refers to a subject exhibiting one or moresymptoms or clinical signs of the condition. “Symptom” refers to anysubjective evidence of disease or of a patient's condition. “Sign” or“clinical sign” refers to an objective physical finding relating to aparticular condition capable of being found by one other than thepatient. As used herein, the term “at risk” refers to a subject that mayor may not actually possess the described risk. Thus, for example, asubject “at risk” of infectious condition is a subject present in anarea where other individuals have been identified as having theinfectious condition and/or is likely to be exposed to the infectiousagent even if the subject has not yet manifested any detectableindication of infection by the microbe and regardless of whether theanimal may harbor a subclinical amount of the microbe.

Accordingly, introduction of the transgenic pathogen can be performedbefore, during, or after the subject first exhibits a symptom orclinical sign of the condition or, in the case of infectious conditions,before, during, or after the subject first comes in contact with theinfectious agent. Treatment initiated after the subject first exhibits asymptom or clinical sign associated with the condition—i.e., therapeutictreatment—may result in decreasing the severity of symptoms and/orclinical signs of the condition, completely resolving the condition,and/or decreasing the likelihood of experiencing clinical evidence ofthe condition compared to an animal to which the composition is notadministered. Similarly, treatment initiated before the subject firstexhibits a symptom or clinical sign associated with the condition—i.e.,prophylactic treatment—may result in decreasing the severity of symptomsand/or clinical signs of the condition, completely resolving thecondition, and/or decreasing the likelihood of experiencing clinicalevidence of the condition compared to an animal to which the compositionis not administered.

The method includes administering an effective amount of the compositionto a subject having, or at risk of having, a particular condition. Inthis aspect of the invention, an “effective amount” is an amounteffective to reduce, limit progression, ameliorate, or resolve, to anyextent, the symptoms or clinical signs related to the condition.

A formulation containing the transgenic pathogen may be provided in anysuitable form including but not limited to a solution, a suspension, anemulsion, a spray, an aerosol, or any form of mixture. The compositionmay be delivered in formulation with any pharmaceutically acceptableexcipient, carrier, or vehicle. The formulation may further include oneor more additives including such as, for example, an adjuvant or aninert carrier (e.g., a nanoparticle), cell culture media, and/orionic/salt solutions.

The amount of transgenic pathogens administered to a subject can varydepending on various factors including, but not limited to, the weight,physical condition, and/or age of the subject, the route ofadministration, the immune status of the subject, and/or the specifictransgenic pathogen. Thus, the absolute amount of transgenic pathogenincluded in a given unit dosage form can vary widely, and depends uponfactors such as the transgenic pathogen, the therapeutic indication, thespecies, age, weight and/or physical condition of the subject, as wellas the method of administration. Accordingly, it is not practical to setforth generally the amount that constitutes an amount of transgenicpathogen effective for all possible applications. Those of ordinaryskill in the art, however, can readily determine the appropriate amountwith due consideration of such factors.

In some embodiments, transgenic pathogen may be administered, forexample, in a single dose to multiple doses. Because the transgenicpathogen can extend the period of an innate immune response and/orgenerate long-lasting T cell responses, it may be possible to administera composition that includes a transgenic pathogen, as described herein,fewer times to control a disease than other conventional therapies.Indeed, the transgenic pathogen can provide an increased innate responsecompared to the innate response generated by therapy that includes acombination of PAMP (or DAMP) and an attenuated form of the pathogenthat is not genetically modified to express the PAMP (or DAMP),regardless of whether the PAMP (or DAMP) is, for example,co-administered with the attenuated form of the pathogen or tethered tothe attenuated form of the pathogen.

In another aspect, this disclosure describes a composition that includesa pathogen that is genetically modified to include a polynucleotide thatencodes a polypeptide involved in innate immunity. The polypeptide caninclude a PAMP or a DAMP as described in detail above.

The transgenic pathogen described herein may be formulated in acomposition along with a “carrier.” As used herein, “carrier” includesany solvent, dispersion medium, vehicle, coating, diluent,antibacterial, and/or antifungal agent, isotonic agent, absorptiondelaying agent, buffer, carrier solution, suspension, colloid, and thelike. The use of such media and/or agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual along with transgenic pathogen withoutcausing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

The transgenic pathogen may be formulated into a pharmaceuticalcomposition. The pharmaceutical composition may be formulated in avariety of forms adapted to a preferred route of administration. Thus, acomposition can be administered via known routes including, for example,oral, parenteral (e.g., intradermal, transcutaneous, intra-peritoneal,subcutaneous, etc.), or topical (e.g., intranasal, intrapulmonary,intramammary, intravaginal, intrauterine, intradermal, transcutaneous,rectally, etc.). For example, a composition can be administered to amucosal surface, such as by administration to, for example, the vaginal,nasal, or respiratory mucosa (e.g., by spray or aerosol). In someembodiments, the composition may be administered orally. In otherembodiments, a composition also can be administered via a sustained ordelayed release.

A formulation may be conveniently presented in unit dosage form and maybe prepared by methods well known in the art of pharmacy. Methods ofpreparing a composition with a pharmaceutically acceptable carrierinclude the step of bringing a transgenic pathogen, as described herein,into association with a carrier that constitutes one or more accessoryingredients. In general, a formulation may be prepared by uniformlyand/or intimately bringing the transgenic pathogen into association witha liquid carrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into the desired formulations.

In the preceding description, particular embodiments may be described inisolation for clarity. Unless otherwise expressly specified that thefeatures of a particular embodiment are incompatible with the featuresof another embodiment, certain embodiments can include a combination ofcompatible features described herein in connection with one or moreembodiments.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Mice, Parasites, and Infections

C57BL/6, B6.IFNγ-knockout (IFNγ^(−/−)), MyD88 Knockout (MyD88^(−/−)),B6.IL-12yet40 reporter (IL-12yet40) and IFNγ reporter (Yeti) mice werepurchased from The Jackson Laboratory (Bar Harbor, Me.) and maintainedin our animal facility under specific pathogen-free conditions. T. cruziepimastigotes were transfected as described previously (Garg et al.,1997. J Immunol 158:3293-3302) with pTREX plasmid (Lorenzi et al., 2003.Gene 310:91-99) containing the coding sequence of Salmonella typhimuriumflagellin (FliC), Neisseria meningitidis (FAM18 strain) porin (PorB), orT. cruzi paraflagellar rod protein 4 (PAR4), with or without fusion toan upstream N-terminal portion of the T. cruzi gp72 gene or influenzahaemagglutinin (HA)-tag, to generate transgenic T. cruzi. All infectionswere initiated by inoculating Vero-cell-culture-passaged trypomastigotestage T. cruzi, intra-peritoneally (i.p) (10⁴ parasites) orsubcutaneously in the ear (s.c) (5×10⁴ parasites) or the foot pad (f.p)(10⁴ parasites). All animal protocols were approved by the University ofGeorgia Institutional Animal Care and Use Committee.

Reporter Cell Assay for NFkB/AP-1 Activation and IL-12 Production

The ability of various T. cruzi strains to induce NFkB/AP-1 activationby TLR stimulation was assayed using THP1-Blue-CD14 reporter cells(InvivoGen, San Diego, Calif.), following the manufacturer's protocol.10⁴ live T. cruzi trypomastigotes were incubated with 2×10⁶ reportercells for nine hours at 37° C./5% CO₂, and the nuclear translocation ofactivated NFkB/AP-1 was determined by colorimetrically quantifying thesecreted embryonic alkaline phosphatase (SEAP). To determine the IL-12production induced in cells by T. cruzi, 10⁵ peritoneal exudatemacrophages from IL-12yet40 reporter mice were incubated with 10³ T.cruzi trypomastigotes for 18 hours at 37° C./5% CO₂. The proportion ofYFP⁺ macrophages was determined by flow cytometry. LPS or media servedas controls.

Determination of Caspase 1 Activity

Active caspases were detected with FLICA Apoptosis Detection kit(ImmunoChemistry Technologies, LLC, Bloomington, Minn.) following themanufacturer's protocol. After 12 hours incubation of 2×10⁴ TcgFliC orTcwt with 2×10⁵ RAW blue (TLR 5) mouse macrophages (InvivoGen, SanDiego, Calif.), the latter were incubated with a fluorescent inhibitorpeptide specific to caspase 1 (FAMYVADFMK, SEQ ID NO:1) for 60 minutesat 37° C./5% CO₂. Inhibitors were removed by rinsing, the cells werefixed, and then the cells were analyzed with a florescence plate reader.

Intracellular Cytokine Staining for IL-1β, IFNγ and TNFα

To measure IL-1β production, 2×10⁵ RAW blue (TLR 5) mouse macrophages(InvivoGen, San Diego, Calif.) were incubated with 2×10⁴ TcgFliCtrypomastigotes for 18 hours. E. coli lipopolysaccharide (LPS)+ATP ormedia served as controls. The induced IL-1β in macrophages weredetermined by staining using the CYTOFIX/CYTOPERM intracellular stainingkit (BD Biosciences, San Jose, Calif.) following the manufacturer'sprotocol.

Similarly, to determine intracellular IFNγ and TNFα production, 1.5×10⁶spleen cells from TcgFliC-infected, TcgPorB-infected, or Tcwt-infected,or naïve mice were restimulated with T. cruzi peptide TSKb20 (5 μM) orT. cruzi whole cell lysate (10 μg) and processed for intracellularcytokine staining (ICS). The splenocytes were washed in PAB (2% BSA,0.02% azide in PBS) and stained for surface expression of CD4, CD44, andCD8 using anti-CD4 PE, CD44 FITC, and anti-CD8 eFluor450 (BDBiosciences, San Jose, Calif.). All cells for ICS were fixed andpermeabilized using CYTOFIX/CYTOPERM (BD Biosciences, San Jose, Calif.)on ice for 15 minutes and washed in PERM/WASH buffer (BD Biosciences,San Jose, Calif.). The cells were then stained with anti-IL-1β PE (R&DSystems, Inc., Minneapolis, Minn.), anti-IFNγ APC or anti-TNFα PECy7(both BD Biosciences, San Jose, Calif.) for 30 minutes on ice. Cellswere washed and fixed in 2% formaldehyde for 20 minutes at 4° C., thenwashed and resuspended in PAB for flow cytometric analysis.

Phenotyping Cells by Flow Cytometry T cell phenotypes were determined asdescribed previously (Bustamante et al., 2008. Nature Med 14:542-550)and stained with tetramer-phycoerythrin (TSKb20-PE; NIH Tetramer CoreFacility) and the following: anti-CD62L APC, anti-CD44 FITC, anti-CD8efluor-450, anti-CD127 PEcy7 and anti-KLRG1 APCcy7 (all fromeBioscience, Inc., San Diego, Calif.). Anti-CD4 PECy5 (Invitrogen, LifeTechnologies Corp., Grand Island, N.Y.) and anti-B220 PECy5 (Invitrogen,Life Technologies Corp., Grand Island, N.Y.) staining was used for adump channel.

To determine the phenotypes of cells infiltrating the site of infection,the tissue (ear) was enzymatically digested to dissociate the cells aspreviously described (Phythian-Adams et al., 2010. J Exp Med207:2089-2096). In the case of draining lymph nodes, the cells weredissociated by gently crushing between the ground edges of glass slides.After FcR (CD16/32) block, cell surface markers were used todifferentiate several cell lineages as previously described(Phythian-Adams et al., 2010. J Exp Med 207:2089-2096). DCs,infiltrating monocytes, or resident macrophage subsets weredifferentiated using the following mAb conjugations: CD11c APC(eBioscience, Inc., San Diego, Calif.), CD8α efluor450 (eBioscience,Inc., San Diego, Calif.), CD11b APC/eFluor780 (BD Biosciences, San Jose,Calif.), Gr-1 (Ly6C/Ly6G) PerCP/Cy5.5 (BioLegend, San Diego, Calif.),F4/80 PECy7 (eBioscience, Inc., San Diego, Calif.) and CD45 PE (BDBiosciences, San Jose, Calif.). DCs were defined with CD11c, withfurther differentiation into CD11b⁻CD8α⁺ cDCs and CD11b⁺ (F4/80⁻) DCs.Monocytes and iDCs were defined as CD11b⁺ CD11c⁻Gr-1^(int) and CD11b⁺CD11c⁺ Gr-1^(int) respectively as described (Turley et al., 2010. NatRev Immunol 10:813-825). Macrophages were identified as F4/80⁺ CD11b⁺CD11c⁻. IL-12 producing DCs were defined as CD11c⁺ CD8α⁺ (CD11b⁻) YFP⁺in IL-12yet40 reporter mice as described before (Reinhardt et al., 2006.J Immunol 177:1618-1627). Data is represented as the percentage of eachcell type over all the cells derived by enzymatic digestion,representing the total cellularity at the site.

IFNγ producing CD8 T cells in the draining lymph nodes of yeti mice weredefined as CD4⁻B220⁻CD8⁺YFP⁺ as described (Mayer et al., 2005. J Immunol174:7732-7739). At least 5×10⁵ (blood) or 5×10⁶ (peripheral tissue/lymphnode) cells were acquired per sample using a CyAn™ flow cytometer(Beckman Coulter, Inc., Brea, Calif.) and analyzed with FlowJo software(Tree Star Inc., Ashland, Oreg.).

Temporary anchoring of PAMPs on T. cruzi

FSL-biotin GPI anchor with a single biotin F-moiety(FSL-CONJ(1Biotin)-5C2-L1, KODE Biotech Materials Ltd., Auckland, NewZealand) was used to coat T. cruzi trypomastigotes according to themanufacturer's protocol. 1×10⁶ trypomastigotes were incubated with 2 μgof FSL-biotin in 100 μl serum free RPMI 1640 media. After washing toremove the excess FSL-biotin, the parasites were incubated on ice withstreptavidin (Sigma-Aldrich, St. Louis, Mo.), at 5× the molarconcentration of FSL-biotin (to give Tc-GPI). Excess streptavidin wasremoved by washing and various biotinylated ligands: FliC-biotin,Pam3CSK4-biotin (Pam3Cys-Ser-(Lys)4-biotin, InvivoGen, San Diego,Calif.), ODN-biotin (oligodeoxynucleotide-biotin, InvivoGen, San Diego,Calif.), all at 3× molar concentration of FSL-biotin or Pam3CSK4-biotinand ODN-biotin together, each at 1.5× molar concentration of FSL-biotinwere incubated with the Tc-GPI for 30 minutes on ice to yieldTc-GPI-FliC, Tc-GPI-Pam3CSK4, Tc-GPI-ODN, or Tc-GPI-ODN-Pam3CSK4,respectively. FliC was biotinylated using EZ-link sulfo-NHS-LCBiotinylation kit (Thermo Fisher Scientific Inc., Rockford, Ill.)following the manufacturer's protocol. The various PAMP-anchored T.cruzi strains were washed twice with RPMI1640 to remove excess PAMPs,counted, re-suspended in complete RPMI1640 and used for in vitro or invivo assays. Tc-GPI was used as the control. PAMPs when co-inoculatedwith T. cruzi, were used at approximately the same quantities (in w/v)as was used to label T. cruzi above.

Statistical Analysis

Data are presented as the mean plus/minus the standard error of mean.Statistical analyses compared the groups with a student t test. Only pvalues of less than 0.05 were considered statistically significant.

Western Blot and ELISA

To determine the presence of FliC, TcgFliC lysate was prepared asdescribed (Martin and Tarleton, 2005. J Immunol 174:1594-1601). Thelysate or culture supernatant (12 hours post-inoculation) were probedwith anti-FliC mAb (BioLegend, San Diego, Calif.) by western blot, asdescribed (Fralish and Tarleton, 2003. Vaccine 21:3070-3080). Seracollected from C57BL/6 mice infected with various T. cruzi strains, 30days post-inoculation were assayed for anti-T. cruzi antibodies by ELISAas described (Gupta and Garg, 2010. PLoS Negl Trop Dis 4:e797).

To determine the relative concentrations of haemagglutinin (HA)-taggedprotein (PAR4-HA) in the trypomastigote stage from various strains oftransgenic T. cruzi, serial dilutions of whole cell lysates were assayedwith anti-HA antibody (Roche). A purified HA-tagged protein (T. cruziPAR2) expressed in E. coli was used as the standard.

Images were acquired with a DeltaVision™ Elite (Applied Precision,Issaquah, Wash.), were deconvolved and adjusted for contrast using itsSoftworx software v5.5 (Applied Precision, Issaquah, Wash.).

Serum Cytokine Assay

Blood collected from C57B1/6 mice inoculated with TcgFliC or Tcwt atfour days post-inoculation and sera separated to assay for variouscytokines using the Q-Plex™ Mouse Cytokine Screen ELISA (QuansysBiosciences, Logan, Utah) following the manufacturer's protocol.Luminescence intensity of each sample was measured and the concentrationof each cytokine was determined using the Q-View™ software (QuansysBiosciences, Logan, Utah) as described (Kriegel and Amiji, 2011. ClinTrans Gastroenterol 2:e2).

Real-Time PCR

The skeletal muscle tissue from mice were analyzed by real-time PCR forthe presence of T. cruzi (DNA) as described before (Cummings, K. L. andTarleton, R. L., 2003. Mol Biochem Parasitol 129:53-59.

Assessment of Infectivity and Clearance of T. cruzi

To assess the infectivity of different strains of T. cruzi, IFNγ^(−/−)mice were inoculated with 10⁴ trypomastigotes of TcgFliC or Tcwtstrains. Blood was collected from the tail vein at 21 dayspost-inoculation to quantify the number of parasites using a compoundlight microscope and expressed as the number of live trypomastigotes per100 (40×) fields. Survival was monitored daily. Clearance of T. cruzifrom infected mice were determined as previously described (Bustamanteet al., 2008. Nature Med 14:542-550).

Determining T. cruzi Specific CD8⁺ T Cell Response and their Phenotypes

The T cell phenotypes were determined as previously described (Kurup, S.P. and Tarleton, R. L., 2013. Nature communications 4:2616). In short,infected or naïve mice peripheral blood was collected by tail bleedingin Alsever's solution (Sigma-Aldrich, St. Louis, Mo.) and washed twicein staining buffer (2% BSA, 0.02% azide in PBS (PAB)). The blood wasthen incubated for 30 minutes at 4° C. in the dark withtetramer-phycoerythrin (PE) and the following antibodies:Allophycocyanin (APC) labeled antibody to CD62L, Fluoresceiniso-thiocyanate (FITC)-labeled antibody to CD44, Allophycocyanin(APC)-Cy7labeled antibody to KLRG1, Phycoerythrin (PE) Cy5-labeledantibody to CD11c, CD4 and B220 for the exclusion channel (all from BDBiosciences, San Jose, Calif.), PE-Cy7 (eBioscience, Inc., San Diego,Calif.) labeled antibody for CD127 and eFluor450-labeled antibody to CD8(eBioscience, Inc., San Diego, Calif.). The stained whole blood was thentreated with hypotonic ammonium chloride solution to lyse red bloodcells, and then washed twice in PAB and fixed using 2% formaldehyde. Weacquired at least 500,000 cells with a CyAn™ flow cytometer (BeckmanCoulter, Inc., Brea, Calif.) and analyzed with FlowJo software (TreeStar Inc., Ashland, Oreg.).

Major histocompatibility complex (MHC) class I tetramers weresynthesized at the Tetramer Core Facility (Emory University, Atlanta,Ga.). The tetramer used in these studies was TSKb20/K^(b) (ANYKFTLV, SEQID NO:2, on H2K^(b)).

The data are represented as percentage of TSKb20/K^(b) tetramer specificCD8⁺ T cells (% TSKb20⁺CD8⁺ T cells) over the total CD8⁺ T cells (FIG.16), or as the proportion of TSKb20/K^(b) tetramer specific, oractivated (CD44⁺) CD8⁺ T cells with the indicated phenotypes (FIGS. 13A,14A, and 17), in circulation in the mice. To determine the fold increasein tetramer, the TSKB20/K^(b) T cell response measured at each timepoint after TcgFliC or Tcwt re-challenge infection in chronicallyinfected mice were divided by their corresponding levels at the time ofre-challenge (FIG. 15).

Determining Tissue Loads of T. cruzi

Tissue loads of T. cruzi were determined as described. (Cummings, K. L.and Tarleton, R. L., 2003. Mol Biochem Parasitol 129:53-59). In brief,mouse skeletal muscle (300 mg) tissues were minced using surgical bladesand added to 5× volume of sodium dodecyl sulfate-proteinase K lysisbuffer containing 10 mM Tris-HCl (pH 7.6), 0.1 M NaCl, 10 mM EDTA, 0.5%sodium dodecyl sulfate and 300 μg of proteinase K/ml. The samples werethen heated for 10 hours at 55° C. and extracted twice withphenol-chloroform-isoamyl alcohol. For quantification, reaction mixturescontained the extracted DNA, 0.5 μM primer mix, 10 μl of iQ™ SYBR GreenPCR Master Mix (Bio-Rad Laboratories, Inc., Hercules, Calif.), andPCR-grade H₂O (Qiagen Inc., Valencia, Calif.) to a final total volume of20 μl. T. cruzi DNA and murine TNF-α DNA (as control for tissue DNAquantity) was amplified using an iCycler iQ™ real-time PCR system(Bio-Rad Laboratories, Inc., Hercules, Calif.). The primers used toamplify T. cruzi DNA were TCSat30: 5′-GGCGGATCGTTTTCGAG-3′ (SEQ ID NO:3)and TCSat179: 5′-AAGCGGATAGTTCAGGG-3′ (SEQ ID NO:4): The T. cruzi loadsin the tissue was represented as DNA equivalents per 50 ng tissue DNA(FIG. 18).

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety. In theevent that any inconsistency exists between the disclosure of thepresent application and the disclosure(s) of any document incorporatedherein by reference, the disclosure of the present application shallgovern. The foregoing detailed description and examples have been givenfor clarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described, for variations obvious to one skilled in the artwill be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

What is claimed is:
 1. A composition comprising a transgenic pathogenthat expresses a heterologous pathogen associated molecular pattern(PAMP) or a damage associated molecular patterns (DAMP).
 2. Thecomposition of claim 1 wherein the PAMP comprises an agonist of at leastone Toll-Like Receptor (TLR).
 3. The composition of claim 2 wherein theTLR comprises TLR 1, TLR 2, or TLR
 5. 4. The composition of claim 1wherein the PAMP comprises an agonist of at least one intracellularpattern recognition receptor (PRR).
 5. The composition of claim 4wherein the PRR comprises Neuronal Apoptosis Inhibitory Protein5/IL-1β-converting enzyme protease-activating factor (NAIP5/Ipaf). 6.The composition of claim 1 wherein the pathogen comprises an infectiouspathogen.
 7. The composition of claim 6 wherein the infectious pathogenis attenuated.
 8. The composition of claim 6 wherein the infectiouspathogen comprises a virus, a bacterium, a fungus, a protozoan, or ahelminth.
 9. The composition of claim 8 wherein the protozoan comprisesT. cruzi.
 10. The composition of claim 1 wherein the pathogen comprisesa tumor cell.
 11. A method of treating an infection in a subject, themethod comprising: administering to the subject, in an amount effectiveto treat the infection, a pathogen genetically modified to express apathogen-associated molecular pattern (PAMP) not natively expressed bythe pathogen.
 12. The method of claim 11 wherein the pathogen comprisesan infectious pathogen.
 13. The method of claim 12 wherein theinfectious pathogen is attenuated.
 14. The method of claim 12 whereinthe infectious pathogen comprises a virus, a bacterium, a fungus, aprotozoan, or a helminth.
 15. The method of claim 14 wherein theprotozoan comprises T. cruzi.
 16. The method of claim 11 wherein thepathogen comprises a tumor cell.